Geographic pattern of genetic variation in Pinus resinosa: area of greatest diversity is not the origin of postglacial populations

Genetic diversity is low in natural populations of red pine, Pinus resinosa, a species that has a vast range across north-eastern North America. In this study, we examined 10 chloroplast microsatellite or simple sequence repeats (cpSSR) loci in 136 individuals from 10 widespread populations. Substantial variation for the cpSSR loci was observed in the study populations. The contrast with red pine's lack of variation for other types of loci is likely to be due to the higher mutation rates typical of SSR loci. The amount of variation is lower than that generally found for cpSSR loci in other pine species. In addition, the variation exhibits a striking geographical pattern. Most of the genetic diversity is among populations, with little within populations, indicating substantial isolation of and genetic drift within many populations in the southern half of the species distribution. The greatest diversity now occurs in the north-eastern part of New England, which is especially intriguing because this entire area was glaciated. Thus the centre of diversity cannot be the origin of postglacial populations, rather it is likely caused by admixture, most probably because of influences from two separate refugia. Furthermore, the pattern indicates that the spread of red pine since the last glaciation is rather more complex than usually described, and it likely includes more than one refugia, complex migration routes, and postglacial-retreat isolation and genetic drift among shrinking populations in regions of the present southern range.     

Extremely low nucleotide polymorphism in Pinus krempfii Lecomte,a unique flat needle pine endemic to Vietnam

Pinus krempfii Lecomte is a morphologically and ecologically unique pine, endemic to Vietnam. It is regarded as vulnerable species with distribution limited to just two provinces: Khanh Hoa and Lam Dong. Although a few phylogenetic studies have included this species, almost nothing is known about its genetic features. In particular, there are no studies addressing the levels and patterns of genetic variation in natural populations of P. krempfii. In this study, we sampled 57 individuals from six natural populations of P. krempfii and analyzed their sequence variation in ten nuclear gene regions (approximately 9 kb) and 14 mitochondrial (mt) DNA regions (approximately 10 kb). We also analyzed variation at seven chloroplast (cp) microsatellite (SSR) loci. We found very low haplotype and nucleotide diversity at nuclear loci compared with other pine species. Furthermore, all investigated populations were monomorphic across all mitochondrial DNA (mtDNA) regions included in our study, which are polymorphic in other pine species. Population differentiation at nuclear loci was low (5.2%) but significant. However, structure analysis of nuclear loci did not detect genetically differentiated groups of populations. Approximate Bayesian computation (ABC) using nuclear sequence data and mismatch distribution analysis for cpSSR loci suggested recent expansion of the species. The implications of these findings for the management and conservation of P. krempfii genetic resources were discussed.     

Molecular and paleo-climatic data uncover the impact of an ancient bottleneck on the demographic history and contemporary genetic structure of endangered Pinus uliginosa

With the current rate of biodiversity loss, conservation management practices require a comprehensive understanding of eco-evolutionary relationships, history, and genetic structure of species. Assessments of genetic diversity are crucial, especially in rare, endemic, or threatened forest tree species with small and isolated populations, such as peat bog pine (Pinus uliginosa N.). Here, we used a novel approach, combining genetic diversity assessment, ecological niche modeling, and population demography inference to explore the complex history of a few remnant populations of this endangered pine. To asses the relative influence of isolation and fragmentation on genetic diversity in the taxonomic context, the patterns of genetic variation found in P. uliginosa were contrasted with those observed in its close relatives with much bigger distribution ranges and larger populations (Pinus sylvestris, Pinus mugo, and Pinus uncinata). We found a similar level of genetic diversity across the species at nuclear loci but contrasting patterns of variability distribution at chloroplast markers. We detected the signatures of an ancient genetic bottleneck dated at around 26 400 years ago, indicating a drastic reduction in the population size of P. uligionosa during the Last Glacial Maximum. In addition, we found substantial differentiation between current populations as a result of enhanced genetic drift during long-lasting isolation. The research suggests potential conservation management strategies for peat bog pine and emphasizes the importance of using complementary approaches for their successful development.     

Comparative genetic study confirms exceptionally low genetic variation in the ancient and endangered relictual conifer,Wollemia nobilis (Araucariaceae)

The Wollemi pine, Wollemia nobilis (Araucariaceae), was discovered in 1994 as the only extant member of the genus, previously known only from the fossil record. With fewer than 100 trees known from an inaccessible canyon in southeastern Australia, it is one of the most endangered tree species in the world. We conducted a comparative population genetic survey at allozyme, amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) loci in W. nobilis, Araucaria cunninghamii and Agathis robusta - representatives of the two sister genera. No polymorphism was detected at 13 allozyme loci, more than 800 AFLP loci or the 20 SSR loci screened in W. nobilis. In Ag. robusta only one of 12 allozyme loci, five of 800 AFLP loci and none of the 15 SSR loci were variable. For A. cunninghamii, 10 of > 800 AFLP loci and five of 20 SSR loci were variable. Thus low genetic diversity characterizes all three species. While not ruling out the existence of genetic variation, we conclude that genetic diversity is exceptionally low in the Wollemi pine. To our knowledge this is the most extreme case known in plants. We conclude that the combination of small population effects, clonality and below-average genetic variation in the family are probable contributing factors to the low diversity. The exceptionally low genetic diversity of the Wollemi pine, combined with its known susceptibility to exotic fungal pathogens, reinforces current management policies of strict control of access to the pines and secrecy of the pine locations.     

Genetic Diversity and Population Structure of Acacia senegal (L) Willd. in Kenya

The level of genetic diversity and population structure of Acacia senegal variety kerensis in Kenya was examined using seven polymorphic nuclear microsatellite loci and two chloroplast microsatellite loci. In both chloroplast and nuclear datasets, high levels of genetic diversity were found within all populations and genetic differentiation among populations was low, indicating extensive gene flow. Analysis of population structure provided support for the presence of two groups of populations, although all individuals had mixed ancestry. Groups reflected the influence of geography on gene flow, with one representing Rift Valley populations whilst the other represented populations from Eastern Kenya. The similarities between estimates derived from nuclear and chloroplast data suggest highly effective gene dispersal by both pollen and seed in this species, although population structure appears to have been influenced by distributional changes in the past. The few contrasts between the spatial patterns for nuclear and chloroplast data provided additional support for the idea that, having fragmented in the past, groups are now thoroughly mixed as a result of extensive gene flow. For the purposes of conservation and in situ management of genetic resources, sampling could target a few, large populations ideally distributed among the spatial groups identified. This should ensure the majority of extant variation is preserved, and facilitate the investigation of variation in important phenotypic traits and development of breeding populations.     

Among population differentiation at nuclear genes in native Scots pine (Pinus sylvestris L.) in Scotland

In the Scottish Highlands, Scots pine is at the north-western extreme of its wide natural distribution. Here, the remaining native populations are patchily distributed in highly variable environments, from the more continental, drier eastern Highlands to the milder, wetter Atlantic Ocean coast. As these pinewoods are the remnants of a naturally established forest, they form a valuable system for analysis of genetic and adaptive variation in heterogeneous environments. Using samples from across the Scottish population, we analysed data from nuclear and mitochondrial genes to assess patterns of within and between population genetic variation. Within population diversity levels were high, and significant genetic differentiation among pairs of Scottish populations at relatively small spatial scales was present at several nuclear loci. At these loci, no differentiation had been found among continental populations, even those separated by large geographic distances. Overall, no clear clustering of Scottish samples was found in population structure analysis suggesting that geographically distant populations with high intra-population nucleotide diversity are not strongly isolated or diverged from each other. Scottish populations lacked a mitotype that is widespread in eastern and north-eastern Europe, indicating that pines from that area may not have participated in the most recent colonisation of the British Isles.     

The importance of reproductive strategies in population genetic approaches to conservation: an example from the marine angiosperm genus Zostera

Knowledge of the levels of genetic diversity maintained in natural populations can play a central role in conservation programmes, particularly in threatened habitats or species. Fluctuations in population size can lead to loss of variation and, consequently, increase the risk of extinction. We have examined whether such a genetic bottleneck has occurred in populations of two species in the seagrass genus Zostera, which are believed to have been affected by an outbreak of wasting disease at the start of the last century. A test for heterozygote excess at five nuclear microsatellite loci did not suggest the occurrence of a genetic bottleneck, but analysis of seven chloroplast microsatellite loci and sequence data from two regions did suggest a bottleneck in the chloroplast genome. Extremely low levels of between-population diversity suggest that all subpopulations can be treated as a single management unit for each species. Comparable levels of nuclear genetic diversity were found in the three populations of the primarily sexual Zostera marina var. angustifolia studied but a wider range of within-population diversity was found in Zostera noltii, which displays both sexual and vegetative reproductive strategies. This may be due to an increase in sexual recruitment due to localised fresh water inflow into the study site near to the most diverse population. Such populations should be prioritised as source material for any replanting or remediation due to natural or anthropogenic loss of Zostera beds in the area.     

Isolation and characterization of polymorphic nuclear microsatellite loci in Pinus cembra L

We developed eight polymorphic nuclear microsatellite markers for the Swiss stone pine (Pinus cembra L.), of which seven may be amplified in a multiplex polymerase chain reaction. Allelic polymorphism across all loci and 40 individuals representing two populations in the Swiss Alps was high (mean = 7.6 alleles). No significant linkage disequlibrium was displayed between pairs of loci. Significant deviation from Hardy–Weinberg equilibrium was revealed at three loci in one population. Cross–amplification was achieved in two related species within the genus (P. sibirica and P. pumila). Thus, the markers may be useful for population genetic studies in these three pine species. They will be applied in ongoing projects on genetic diversity and patterns of gene flow in P. cembra.     

Genetic variation at allozyme and RAPD loci in sessile oak Quercus petraea (Matt.) Liebl.: the role of history and geography

The nuclear genetic variation within and among 21 populations of sessile oak was estimated at 31 RAPD loci in conjunction with previous estimates of variation at eight allozyme loci. The aim of the study was to assess the relative role of isolation-by-distance and postglacial history on patterns of nuclear variation. Because of its small effective population size and maternal transmission, the chloroplast genome is a good marker of population history. Both kinds of nuclear variation (RAPD and allozyme) were therefore compared, first, to the geographical distances among populations and, secondly, to chloroplast DNA restriction polymorphism in the same populations. Multiple Mantel tests were used for this purpose. Although RAPDs revealed less genetic diversity than allozymes, levels of genetic differentiation ( G _ST) were identical. The standard genetic distance calculated at all RAPD loci was correlated with geographical distances but not with the genetic distance calculated from chloroplast DNA data. Conversely, allozyme variation was correlated with chloroplast DNA variation, but not with geography. Possibly, divergent selection at two allozyme loci during the glacial period could explain this pattern. Because of its greater number of loci assayed, RAPDs probably provided a less biased picture of the relative role of geography and history.     

Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga.     

Structure of Pinus nigra Arn. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis: Provenance tests

Pinus nigra is a forest and low elevation mountain species found around the Mediterranean Sea that has had its distribution reduced and fragmented by anthropogenic disturbance. Due to commercial interest it is currently being replanted, however, the genetic structure of populations is little known and current planting strategies could threaten its genetic diversity. In the present study we investigated the genetic structure and genetic diversity of P. nigra populations in Bulgaria using chloroplast microsatellite markers and terpene analysis. Nine provenances were chosen throughout the species' range in Bulgaria. Following DNA extraction, chloroplast microsatellite (cpSSR) loci were surveyed using three primer pairs. Between 5 and 9 size variants were identified at each locus. A total of 22 size variants at the 3 loci were identified, that were combined in 68 different haplotypes, of which 7 represent 39.81% of the genetic structure. AMOVA analysis revealed that 6.06% of the variation was found among populations, while 93.94% was expressed within populations. The cpSSR analysis divided European Black pine populations into four groups, the first represented by populations located the eastern Rhodopes, Sr. Gora and St. Planina mountains, while the second group is primarily located in the Phodopes and Slavianca mountains. The populations from Pirin and Osogovo mountains show different genetic patterns. Terpene analysis revealed that most of the monoterpene pool in P. nigra was accounted for by α-pinene followed by β-pinene. The presence of four distinct terpene groups is not consistent with physical distances between populations, and a similar non-significant correlation between genetic distance determined by chloroplast microsatellites analysis and chemotype distance (determined by terpenes) was observed. Our results suggest that the structural pattern of genetic diversity of cpDNA in European Black pine populations is the consequence of historical biogeographic processes.     

Nuclear microsatellites for Pinus pinea (Pinaceae), a genetically depauperate tree, and their transferability to P. halepensis

? Premise of the study: Pinus pinea is one of the few widespread plant species that are also genetically depauperate. It is also an important commercial species with high market value seeds. A deeper knowledge of the existing population genetic variation was needed. ? Methods and Results: Twelve nuclear microsatellites were isolated from genomic and cDNA sequences and screened for variability in 729 individuals from 33 natural populations. Low level of genetic variability was confirmed with average expected heterozygosity of 0.11. Hardy-Weinberg equilibrium expectations were not met in only ～10% of the possible locus/population combinations. All loci were in linkage equilibrium, and the frequency of null alleles was very low (≤1% in 332 out of 396 locus/population combinations). Nine out of the 12 microsatellites were successfully transferred to P. halepensis. ? Conclusions: Despite low polymorphism, these new markers will be useful to resolve population structure and hold potential for seed origin identification and traceability.     

马尾松和黄山松两个核基因位点的群体遗传多样性和种间分化

利用两个核基因座位C3H和GI, 对重叠分布于中国东南部的两个松属(Pinus)物种马尾松(P. massoniana)和黄山松(P. hwangshanensis)的22个群体88个个体进行了遗传多样性和种间分化模式研究。在这两个核基因座位上, 两种植物都表现出较低的核苷酸多样性水平(马尾松π_sil = 0.001 71; 黄山松π_sil = 0.003 40), 但是马尾松要显著低于黄山松; 在种内分化水平上, 马尾松的种内遗传分化也明显低于黄山松(马尾松F_ST = 0.059; 黄山松F_ST = 0.339)。这可能是由于黄山松的海拔分布高于马尾松, 而高海拔分布使黄山松的分布区域更加片段化, 促使其形成较高的种内遗传多样性和遗传分化。分子变异分析(AMOVA)发现, 两物种基于两个核基因座位的种间差异为48.86%, 而GI基因座位上的种间差异明显高于C3H座位(GI: 77.24%, C3H: 20.48%), 同时, 基因谱系显示两物种的共享单倍型仅在C3H座位上存在。结合这两个基因的功能, 推测GI基因可能在物种形成过程中受到了一定的选择压力, 因为GI基因参与调控植物的开花时间, 而C3H与木质素表达水平的调控有关。不同的选择压力使得GI的进化速度相对较快, 从而加速了黄山松和马尾松的物种分化。     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

Tracking a genetic signal of extinction-recolonization events in a neotropical tree species: Vouacapoua americana Aublet in French Guiana

Drier periods from the late Pleistocene and early Holocene have been hypothesized to have caused the disappearance of various rainforest species over large geographical areas in South America and restricted the extant populations to mesic sites. Subsequent improvement in climatic conditions has been associated with recolonization. Changes in population size associated with these extinction-recolonization events should have affected genetic diversity within species. However, these historical hypotheses and their genetic consequences have rarely been tested in South America. Here, we examine the diversity of the chloroplast and nuclear genomes in a Neotropical rainforest tree species, Vouacapoua americana (Leguminosae, Caesalpinioideae) in French Guiana. The chloroplast diversity was analyzed using a polymerase chain reaction-restriction fragment length polymorphism method (six pairs of primers) in 29 populations distributed over most of French Guiana, and a subset of 17 populations was also analyzed at nine polymorphic microsatellite loci. To determine whether this species has experienced extinction-recolonization, we sampled populations in areas supposedly not or only slightly affected by climatic changes, where the populations would not have experienced frequent extinction, and in areas that appear to have been recently recolonized. In the putatively recolonized areas, we found patches of several thousands of hectares homogeneous for chloroplast variation that can be interpreted as the effect of recolonization processes from several geographical origins. In addition, we observed that, for both chloroplast and nuclear genomes, the populations in newly recolonized areas exhibited a significantly smaller allelic richness than others. Controlling for geographic distance, we also detected a significant correlation between chloroplast and nuclear population differentiation. This result indicates a cytonuclear disequilibrium that can be interpreted as a historical signal of a genetic divergence between fragmented populations. In conclusion, the spatial genetic structure of contemporary V. americana populations shows evidence that this species has experienced large extinction-recolonization events, which were possibly caused by past climatic change.     

Genetic structure of a regionally endangered orchid,the dark red helleborine (<Emphasis Type="Italic">Epipactis atrorubens</Emphasis>) at the edge of its distribution

The genetic structure and diversity of species is determined by both current population dynamics and historical processes. Population genetic structure at the edge of the distribution is often expected to differ substantially from populations at the centre, as these edge populations are often small and fragmented. In addition, populations located in regions that have experienced repeated glaciations throughout the Pleistocene, may still carry imprints from the genetic consequences of frequent distribution shifts. Using chloroplast DNA sequences and nuclear microsatellite markers we studied the genetic structure of Epipactis atrorubens at the northern edge of its distribution. Contrary to populations in the centre of the distribution, populations at the northern range are regionally endangered as they are small and disjunct. Sequence data of 2 chloroplast loci and allelic data from 6 nuclear microsatellite markers were obtained from 297 samples from Finland, Estonia and Russia. We sought for genetic indicators of past population processes, such as post-glacial colonisation history of E. atrorubens. As expected, we observed low genetic variation, in terms of numbers of substitutions, haplotypes and alleles, and significant levels of differentiation, especially pronounced in the chloroplast DNA. These features suggest that the edge populations could be prone to extinction.     

Testing Putative African Tropical Forest Refugia Using Chloroplast and Nuclear DNA Phylogeography

The location and definition of Pleistocene refugia for tropical forest assemblages remains controversial. Phylogeographic methods have been used successfully in temperate ecosystems to locate past forest refugia using genetic data that coincide with pollen core evidence, and in some cases provide the sole basis for their location. Here we present molecular phylogeographic data from nuclear and chloroplast loci for the forest tree Irvingia gabonensis, across the majority of its natural range, in Nigeria, Cameroon and Gabon. It is the first detailed phylogeographic study to posit the location of tropical forest refugia across this region of Africa. Using the same method of restriction fragment length polymorphism screening, 17 alleles were identified across five anonymous nuclear loci and two haplotypes at a single chloroplast locus. Analysis based on nuclear variation identified two genetically diverse, differentiated allelic clusters within the species range, one in southern Nigeria/western Cameroon and the other in southwestern Cameroon. Molecular data are consistent with a historical genetic contraction and bottleneck into these regions in the Pleistocene and/or Holocene, which has been followed by subsequent expansion. Both genetic refugia are located within areas previously suggested as forest refugia from biogeographic studies, supported by available pollen core data, and occur either side of the Sanaga River, a notable biogeographic divide for mammals (particularly primates). Other putative refugia in Gabon do not appear to have acted as genetic refugia for I. gabonensis, and Gabon was most likely recolonised from the SW Cameroon refugial source. In this study, nuclear loci were able to highlight significant phylogeographic structure across the range of a tropical African tree, whereas chloroplast analysis gave a much more limited picture. With the increased availability of sequence data for non-model species, the de novo development and further application of nuclear loci is strongly recommended for phylogeographic studies of plants.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.